Project Summary: The long-term goal of this project is to elucidate the impact of the transient receptor potential (TRP) superfamily of cation channels in the control of vascular function. In this competitive renewal application, we propose a series of novel studies designed to ascertain the role of the TRP mucolipin 1 (TRPML1) channel in the regulation of vascular smooth muscle cell (SMC) contractility and to investigate the pathological involvement of the channel during systemic hypertension. TRPML1 is a Ca2+-permeable cation channel primarily localized to the membranes of late endosomes and lysosomes. Activation of the channel results in release of Ca2+ into the cytosol. We put forth the novel mechanistic hypothesis that TRPML1 forms a Ca2+-signaling complex with type-2 ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) and large conductance Ca2+-activated K+ (BK) channels on the plasma membrane. Prior studies have shown that release of Ca2+ through RyR2 generates transient subcellular Ca2+ signals known as ?Ca2+ sparks?. A single Ca2+ spark activates multiple BK channels, stimulating large K+ currents that hyperpolarize and relax SMCs to cause vasodilation. Our new model proposes that TRPML1 is indispensable for the initiation of this critical pathway, suggesting a previously unknown, yet vital, role for the channel in vascular control. We further propose that uncoupling of TRPML1 from RyR2 during hypertension underlies vascular pathologies associated with the disease. The goal of Aim 1 is to elucidate the signaling pathways regulated by TRPML1 channels that control SMC contractility. These studies will develop and utilize novel genetically encoded Ca2+ biosensors targeted to endosomes to optically record TRPML1 activity in SMCs. Super-resolution microscopy will be used in conjunction with selective pharmacological activators, patch-clamp electrophysiology, and pressure myography to test the hypothesis that TRPML1 and RyR2 form a closely coupled Ca2+-signaling complex in SMCs that is essential for vasodilation via the Ca2+ spark/BK pathway. Because physiological regulation of TRPML1 activity is poorly understood, the goal of Aim 2 is to test the hypothesis that endogenous generation of reactive oxygen species (ROS) regulates TRPML1 activity in SMCs. Super-resolution microscopy, newly developed endosomal Ca2+ biosensors, and DHE ROS imaging will be used to test the hypothesis that TRPML1 co-localizes with NADPH oxidase 2 (NOX2) and that ROS generated by NOX2 regulate TRPML1 activity in SMCs. The goal of Aim 3 is to elucidate the pathological involvement of TRPML1 channels during systemic hypertension. We hypothesize that pathological increases in ROS generation associated with hypertension uncouple TRPML1 from RyR2, thereby disabling the Ca2+ spark/BK pathway and increasing vasoconstriction. We further propose that uncoupled TRPML1 Ca2+ signals contribute to SMC proliferation and migration associated with vascular remodeling. All Aims will utilize resistance arteries from wild-type and TRPML1-knockout mice. Parallel studies using small arteries recovered from human donors increase impact and significance.